Integrated Regulation of Signal Coding and Plasticity by NMDA Receptors at a Central Synapse

The role of NMDA and non-NMDA glutamate receptors in long-term potentiation has been intensely investigated, yet recent evidence on the dynamics of synaptic depolarization suggests that the original view should be extended. NMDA receptor-mediated currents, apart from their Ca2+ permeability, show a marked voltage dependence, consisting of current increase and slowdown during membrane depolarization. During highfrequency synaptic transmission, NMDA current increase and slowdown are primed by non-NMDA receptor-dependent depolarization and proceed regeneratively. Thus, NMDA receptors make a decisive contribution to membrane depolarization and spike-firing. From the data obtained at the mossy fiber- granule cell synapse of the cerebellum, we propose that the electrogenic role of NMDA receptors is functional to LTP induction. Moreover, during LTP, both NMDA and non- NMDA receptor currents are potentiated, thus establishing a feed-forward mechanism that ultimately enhances spike firing. Thus, NMDA receptors exert an integrated control on signal coding and plasticity. This mechanism may have important implications for information processing at the cerebellar mossy fibergranule cell relay.

glutamate receptors in long-term potentiation has been intensely investigated, yet recent evidence on the dynamics of synaptic depolarization suggests that the original view should be extended. NMDA receptor-mediated currents, apart from their Ca 2+ permeability, show a marked voltage dependence, consisting of current increase and slowdown during membrane depolarization. During highfrequency synaptic transmission, NMDA current increase and slowdown are primed by non-NMDA receptor-dependent depolarization and proceed regeneratively. Thus, NMDA receptors make a decisive contribution to membrane depolarization and spike-firing.
From the data obtained at the mossy fibergranule cell synapse of the cerebellum, we propose that the electrogenic role of NMDA receptors is functional to LTP induction. Moreover, during LTP, both NMDA and non-NMDA receptor currents are potentiated, thus establishing a feed-forward mechanism that ultimately enhances spike firing. Thus, NMDA receptors exert an integrated control on signal coding and plasticity. This mechanism may have important implications for information processing at the cerebellar mossy fibergranule cell relay. INTRODUCTION Synaptic transmission at glutamatergic synapses of the vertebrate central nervous system is brought about by the activation ofN-methyl-Daspartate (NMDA) and non-NMDA receptors. In the past decade, several investigations have addressed the biophysical and molecular aspects of glutamatergic transmission (for review, see McBain, 1994;Edmonds et al., 1995;Kaczmarek et al., 1997). In particular, the relevance of glutamate-channel properties to synaptic plasticity [long-term potentiation (LTP) and long-term depression (LTD)] has been revealed (Bliss & Collingridge, 1993;Bear & Malenka, 1994). Yet, recent evidence on the dynamics of synaptic depolarization suggests that an extension of the original view is needed.

FUNCTIONAL PROPERTIES OF NMDA RECEPTORS
NMDA receptors show complex functional properties. NMDA receptors are associated with an ionic channel that is blocked by Mg 2+ at negative membrane potentials (Nowak et al., 1984;Mayer et al., 1984). Once unblocked, the NMDA channel is highly permeable to Ca 2+ (Mayer & Westbrook, 1987;Iino et al., 1990).
The permeability allows a synaptic regulation of Ca + influx, which is of fundamental importance  Silver et al., 1992;D'Angelo et al., 1993). (C) The NMDA current can be fitted by a double exponential function (individual exponential curves are shown as broken lines). Voltage-dependence of the two decay time constants is reported in a semi-logarithmic plot for three granule cells. Data partly from D'Angelo et al. (1993,1994).
for the induction of synaptic plasticity (Bliss & Collingridge, 1993;Bear & Malenka, 1994). A second property that has been less considered so far is that NMDA receptor-mediated synaptic currents slow down during membrane depolarization (Keller et al., 1991).
On the basis of experiments carried out at the mossy fiber-granule cell synapse of the cerebellum, we propose that NMDA current voltagedependent kinetics are also functional to LTP induction.

WHOLE-CELL RECORDINGS FROM GRANULE CELLS IN RAT CEREBELLAR SLICES
The results reported in this paper extend our previous work on rat cerebellar granule cells (D'Angelo et al., 1993(D'Angelo et al., -19951997a;Rossi et al., 1996; see also Silver et al., 1992). The present experiments were performed using the whole-cell configuration of the patch-clamp in cerebellar slices (Edwards et al., 1989). Synaptic currents were elicited by electrical stimulation of the mossy fiber bundle. Owing to the simplified organization of their dendritic structure and mossy fiber input, cerebellar granule cells are electrotonically compact, and a direct relationship between synaptic current properties and effects on neuronal excitability can be established.

NMDA RECEPTOR-DEPENDENT REGULATION OF SYNAPTIC TRANSMISSION
During low-frequency transmission, non-NMDA receptors are almost entirely responsible for synaptic depolarization (Collingridge et al., 1988a,b;Collingridge, 1992). Nonetheless, a contribution of NMDA receptors was revealed ( Fig. 2A) in the large-size excitatory postsynaptic potentials (EPSP) of cerebellar granule cells, in which the Mg 2/ block is removed, and the NMDA current slows down. In these conditions, the NMDA current increases the EPSP amplitude and protracts EPSP decay (D'Angelo et al., 1995). An NMDA receptor contribution to low-frequency EPSPs is particularly evident in immature synapses, which show a high NMDA/non-NMDA current ratio (D'Angelo et al., 1993(D'Angelo et al., , 1997a. A critical contribution of the NMDA current occurs in cerebellar granule cells during highfrequency synaptic activity (Fig. 2B), when the NMDA current sustains membrane depolarization and spike firing (D'Angelo et al., 1995). Apparently, the role of non-NMDA currents is to prime the regenerative cycle protracting NMDA current duration. Non-NMDA currents, however, undergo a strong depression during the burst, so that their direct contribution to depolarization is small. Non-NMDA current depression is probably due to channel desensitization (Tmssel et al., 1993;Silver et al., 1996), whereas NMDA current desensitization is prevented by glycine, a coagonist of glutamate at the NMDA receptor (Mayer et al., 1989).
It turns out that, during repetitive highfrequency synaptic transmission, the NMDA current greatly enhances synaptic depolarization and spike firing (Fig. 2C). This effect is important because repetitive high-frequency burst firing is a common mode of information coding in neuronal networks, simultaneously providing an adequate stimulus for the induction ofLTP.

NMDA RECEPTOR-DEPENDENT REGULATION OF SYNAPTIC PLASTICITY
The best known form of synaptic plasticity is LTP, a candidate for the deposition of memory in neuronal circuits (Bliss & Collingridge, 1993).
LTP consists of a brief induction phase, driven by bursts of high-frequency synaptic impulses or by various pairing procedures, followed by a longlasting expression phase, during which synaptic After induction, both the non-NMDA current (peak amplitude at-70 mV) and the NMDA current (dotted line at +60 mV) were increased. Potentiation was induce by (A) low-frequency NMDA receptor synaptic activation (0.1 Hz) paired with a 60-see application of the metabotropic glutamate receptor agonist 20 glVl t-ACPD, or by 03) high-frequency NMDA receptor synaptic activation (8 bursts of 10 impulses at 100 Hz every 250 ms) paired with membrane depolarization to -40 mV. (C) Subtraction of control from potentiated tracings at +60 mV showed that the NMDA current increased in a slow component (same cell as in B). Data in A obtained from Rossi et al., 1996. transmission is potentiated (Fig. 3 A,B). As at other glutamatergic synapses, LTP induction at the mossy fiber-granule cell synapse depends on NMDA receptors D'Angelo et al., 1997b;D'Angelo et al., unpublished;Fig. 3C). NMDA receptors, by causing Ca 2+ influx through the associated channel, activate the intracellular Ca2+-dependent mechanisms leading to LTP. To unblock NMDA channels from Mg2/, the postsynaptic neuron must be depolarized.
As at other glutamatergic synapses (see also O'Connor et al., 1995;Clark & Collingridge, 1995;Kullman et al., 1996), during LTP expression at the mossy fiber-granule cell synapse, both the NMDA and non-NMDA are potentiated. Moreover, the NMDAcurrent slows down considerably (Fig. 3C), apparemly in relation with a selective increase in the slow component of NMDA-EPSC decay . INTEGRATED Fig. 4: Integrated regulation of signal coding and plasticity by NMDA receptors. In this model, NMDA-receptor activation is critical for both membrane electrogenesis and Ca / influx. At the core of this process remains a voltage-dependent increase in size and duration of the NMDA current during membrane depolarization, with non-NMDA receptors priming the regenerative process. The NMDA receptors, by increasing their own responsiveness, configure the system in feed-forward mode. Note that the final effect will be an enhancement of repetitive firing, and therefore of the input-output function of the neuron.

REPETITIVE FIRING
At the core of this process is the voltagedependent increase in the size and duration of the NMDA current during membrane depolarization. Non-NMDA receptors would have the important role of priming the regenerative NMDA current increase. Once LTP is established, the increase in non-NMDA and NMDA currents, and the slowdown in NMDA current, enhance membrane depolarization and spike firing. In the model proposed in Fig. 4, NMDA receptors increase their own responsiveness after high-frequency transmission. Thus, the system is configured in feed-forward mode, rendering the potentiated synapses particularly susceptible to develop further LTP (Bortolotto et al., 1994). In practice, once the induction threshold is crossed, LTP should tend toward saturation. Although a feedforward mechanism is attractive because it ensures a high reliability for LTP induction, such a mechanism intrinsically requires a negative control. Beside numerous LTP modulatory mechanisms, GABA-receptor-mediated synaptic inhibition is an interesting candidate as, by preventing membrane depolarization, it would break down the electrogenic mechanism that is based on regenerative NMDA current activation. It should be noted that most synaptic inhibition may be effective at low frequency, but be turned off by GAB A autoreceptors at high-frequency.
A WIDESPREAD PROPERTY OF GLUTAMATERGIC SYNAPSES?
The model proposed in Fig. 4 is largely based on observations on mature cerebellar granule cells, in which NMDA receptors express the NR2-A and NR2-C subunits (Ebralidize et al., 1996;Takahashi et al., in press). Regional and ontogenetic differences in NMDA-receptor subunits affect NMDA receptor properties (see Monyer et al., 1994). We present evidence below, however, suggesting that an integrated NMDA receptor-dependent control of synaptic transmission and plasticity may be effective in different brain areas.
In hippocampal synapses, the NMDAreceptor-mediated synaptic current and its role in synaptic plasticity have been very extensively characterized. At the perforant path-granule cell synapse, NMDA receptors contribute to synaptic transmission (Lambert & Jones, 1990), and the NMDA current shows marked voltage-dependent kinetics (Keller et al., 1991). Moreover, at this synapse, NMDA receptors are both required for LTP induction and potentiated during LTP expression (O'Connor et al., 1995). The perforant path-granule cell synapse is, therefore, a good candidate for an integrated NMDA-receptormediated control of synaptic transmission and plasticity. At the Shaffer collateral-CA1 synapse, NMDA receptors are likewise involved in both LTP induction and expression (Clark & Coilingridge, 1995). Nevertheless, NMDA current voltage dependence is weak (Hestrin et al., 1990), and synaptic transmission is largely sustained by non-NMDA receptors (Collingridge et al., 1987a,b). The applicability of our model to the Shaffer collateral-CA1 synapse is less clear than it is to the perforant path-granule cell synapse.
Functional evidence for a dual role of.NMDA receptors in signal coding and synaptic plasticity is found in the visual system. In the lateral geniculate nucleus, NMDA receptors sustain high-frequency transmission of visual information (Heggelund & Hartveit, 1990;Turner et al., 1994). In the visual cortex, NMDA receptors are necessary to both mediate the visual response (Miller et al., 1989) and generate LTP (Artola & Singer, 1992). Interestingly, ocular dominance plasticity requires NMDA-receptor activation during normal synaptic transmission (Kasamatsu et al., 1998). Thus, the functional role of NMDA receptors in the visual system is compatible with the model proposed here. CONCLUSIONS By including the dynamics of postsynaptic membrane depolarization during repetitive highfrequency synaptic transmission into the previous knowledge on LTP, we have extended the functional implications of NMDA receptors. NMDA receptors exert an integrated control on signal coding and plasticity, which is especially relevant during repetitive high-frequency synaptic transmission. This mechanism may have important implications for information processing at the cerebellar mossy fiber-granule cell relay, as well as at other central synapses.